Quantification of threonine, serine and tyrosine phosphorylation in TLR signaling pathways. The TLRs are a family of pathogen recognition receptors that alert the host to the presence of pathogens by recognizing molecular signatures, termed pathogen-associated molecular patterns (PAMPs). These sensors act as the first step in the induction of protective innate and adaptive immune responses. There are 11 human TLR homologues and they are each activated by one or more PAMP ligands. TLRs are all transmembrane proteins and their signaling is mediated by association of their internal domains with intracellular components. Classically, the TLR signaling cascade involves the myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-activated kinase (IRAK), and tumor-necrosis factor receptor-associated factor 6 (TRAF6), leading to the activation of Nuclear Factor kappaB (NF-kB). Among the most important genes to be regulated by TLR signaling are those encoding cytokines. Given the key role of cytokines in the orchestration of the inflammatory response, mechanisms of modulating their production has garnered substantial interest, in particular in the area of the development of therapies for the treatment of chronic inflammatory diseases. A clearer understanding of the TLR pathway leading to the cytokine production is required for a successful pharmacological intervention. A) We investigated differences in the phosphoprotein signaling cascades triggered by TLR2, TLR4, and TLR7 ligands using as a responding population a well-characterized murine macrophage cell line. We performed a global, quantitative, early poststimulation kinetic analysis of the mouse macrophage phosphoproteome using stable isotope labeling with amino acids coupled to phosphopeptide enrichment and high-resolution mass spectrometry. For each TLR ligand, we found marked elevation of phosphorylation of cytoskeleton components, GTPases of the Rho family, and phospholipase C signaling pathway proteins. Phosphorylation of proteins involved in phagocytosis was only seen in response to TLR2 and TLR4 but not to TLR7 activation. Changes in the phosphorylation of proteins involved in endocytosis were delayed in response to TLR2 as compared to TLR4 ligands. These findings reveal that the phosphoproteomic response to stimulation of distinct TLRs varies both in the major modification targets and the phosphorylation dynamics. These results advance the understanding of how macrophages sense and respond to a diverse set of TLR stimuli. The data were deposited in the publicly available online database Proteome Exchange. We will use the phosphorylation dataset as additional constraints for a computational model of the TLR signaling network. The candidate proteins whose phosphorylation changed significantly during the investigated time course are being further examined in follow up biological experiments. B). We are optimizing another relative quantification method based on post-processing peptide labeling - the dimethylation method, which should be extremely useful for proteomic and phosphoproteomic studies of primary cells and tissues not amenable to the SILAC protocol. The method was tested on murine macrophage lysates and initial results were compared to the SILAC- quantified study. The dimethyl labeling will be applied to studies of the TLR phosphoproteome in primary murine macrophages. C). To complete the TLR signaling network and assess the proteome dynamics, we are conducting parallel studies of the proteome and secretome changes using the same cells and ligands as for the phosphoproteome analysis, but collecting data after longer periods of time to allow for changes in protein expression and secretion The data will provide more stringent constraints for the TLR signaling model. 2. T-cell receptor signaling - quantification of tyrosine phosphorylation. Recognition of foreign antigens by T lymphocytes is an important step in the initiation of the immune response. Unlike growth factor receptors, the TCR doesn't have intrinsic enzymatic activity. Upon foreign antigen recognition via MHC/TCR, Src family phosphotyrosine kinase (PTK) activity results in immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation and recruitment of ZAP-70 PTK. This allows the TCR complex to function as an active PTK. ZAP-70 phosphorylates adaptor proteins such as LAT and SLP-76 results in activation of phospholipase C (PLC) leading to Ras/Raf1/ERK activation and Ca++ flux. We use T lymphocytes isolated from the 5C.C7 mouse strain and activated in vitro with the antigen presenting cells (line P13.9) as the experimental model system. We have obtained initial qualitative data in this project. 3. Analysis of post-translational modifications of CENP-A (collaboration with Yamini Dalal, NCI): In eukaryotes DNA is packaged into chromatin by essential histone proteins. Specialized histone variants such as centromere-specific histone H3 (CENP-A) provide a structural and epigenetic basis for chromosome segregation by marking centromeres. To maintain centromere parity after replication, CENP-A must segregate equally to nascent daughter DNA strands. How cells prevent unequal distribution of CENP-A to daughter strands after replication fork passage is unknown. We have characterized novel modifications within the histone fold domains of CENP-A and H4 that occur at G1-S cell cycle transition, which coincide with loss of the chaperone HJURP binding, suggesting a mechanistic basis for CENP-A structural conversion. We have established that accurate post-translational modification mapping is possible from minute amounts of protein, using ultra-sensitive mass spectrometry, and bioinformatic tools combined with careful manual spectra validation. We are continuing the detailed characterization of the modification states of CENP-A dependent on the cell cycle stage. Time-resolved, stimulus-dependent quantitative changes of phosphorylation of individual sites are being obtained using sensitive nanospray-based mass spectrometry (LTQ Orbitrap Velos from Thermo) combined with Single Reaction Monitoring. The dynamic modification of selected individual phosphorylation sites is being confirmed by Western blotting. We are now using TAU gels for the optimal separation of histone variants to obtain comprehensive modification map of CENP-A. The TAU gels and mass spectrometry are a novel combination of methods for histone post-translational modification analysis. 4. We are conducting a quantitative analysis by mass spectrometry (MS) of the phosphoproteome changes in the myeloma cells treated with rapamycin and/or with entinostat, using SILAC labeling with a specific focus on the mTOR signaling pathway. We have obtained interesting preliminary results that suggest potential biologically significant phosphoproteome changes in response to drug treatment at one time point and we will follow up with more fine-grained, comprehensive analyses. References: 1. Sjoelund V, Smelkinson M, and Nita-Lazar A. (2014) Phosphoproteome Profiling of the Macrophage Response to Different Toll-Like Receptor Ligands Identifies Differences in Global Phosphorylation Dynamics. J. Proteome Res. DOI: 10.1021/pr5002466